Thymocyte-Dendritic Cell Interactions near Sources of CCR7 Ligands in the Thymic Cortex

Abstract

Little is known about the dynamics of the interactions between thymocytes and other cell types, as well as the spatiotemporal distribution of thymocytes during positive selection in the microenvironment of the cortex. We used two-photon laser scanning microscopy of the mouse thymus to visualize thymocytes and dendritic cells (DCs) and to characterize their interactions in the cortex. We show that thymocytes make frequent contacts with DCs in the thymic cortex and that these associations increase when thymocytes express T cell receptors that mediate positive selection. We also show that cortical DCs and the chemokine CCL21 To what extent the cortex itself is divided into distinct functional compartments is not yet clear. At one end of the spectrum, it is possible that the cortex is functionally homogeneous. According to this view, cortical thymocytes would invariably be in contact with thymic epithelial cells capable of mediating positive selection, and positive selection could take place with equal probability throughout the cortex. On the other hand, there are indications that cortical thymic epithelial cells are heterogeneous (reviewed in Ref. 7), raising the possibility of functionally distinct regions of the cortex specialized for mediating selection events. The anatomical segregation of thymocytes in the cortex based on their TCR specificity provides an indication for such heterogeneity (8). However, distinct functional regions of the thymic cortex have not yet been identified. Two-photon laser scanning microscopy (TPLSM) provides an opportunity to examine the cellular migration and interaction events in tissues that underlie selection events within the thymus (reviewed in Ref. 9). Initial studies have revealed that thymocytes undergo a calcium-dependent stopping in response to positive selection signals in the thymus (10) and that interaction of thymocytes with MHC-bearing stromal cells can lead to prolonged but dynamic cell-cell contacts (11). Additionally, analysis of cell migration in the cortex of intact thymic lobes provided evidence that thymocytes move via a random walk in the cortex before positive selection and undergo rapid, directed migration to the medulla as a result of positive selection (12). These studies provide an early glimpse into the regulation of thymocyte migration by positive selection. However, the interactions of thymocytes with other cell types in the intact thymus and how these interactions change during the process of positive selection have not yet been addressed.Herein we examine the impact of the chemokine receptor CCR7 and TCR repertoire selection on thymocyte-DC interactions in the cortex of intact thymic lobes. We show that thymic DCs form intimate associations with cortical capillaries near sources of CCR7 ligands in the cortex. We find that thymocytes extensively

Opposing chemokine gradients control human thymocyte migration in situ

“…Immature DN and DP thymocytes in the cortical region of mouse thymic slices migrated with speeds of approximately 4 μm/min, similar to previous reports for mouse cortical DN and polyclonal DP thymocytes from both intact and vibratome-cut thymic lobes (27,29,30). Mature human CD4 + and CD8 + SP thymocytes in the medulla migrated more rapidly than did immature cells in the cortex, exhibiting average speeds of approximately 9 and 12 μm/min, respectively ( Figure 2, A and B, and Supplemental Video 1; supplemental material available online with this article; doi:10.1172/ JCI67175DS1).…”

“…In fact, as reported here and shown previously with mouse thymocytes (8,27,29), cortical thymocytes were more than 2-fold slower than their medullary counterparts. Moreover, we found that PTX treatment sharply reduced the speed of human SP thymocytes, but had a minor effect on DP thymocyte speed.…”

“…Although positive selection is thought to require specific recognition of peptide-MHC complexes on thymic stromal cells, there are also indications that a large proportion of DP thymocytes receive low-level TCR signals from MHC recognition, leading to upregulation of activation markers and maintenance of basal motility in the thymic cortex (29,33,34). To determine whether mouse MHC can provide these signals to human thymocytes, we cultured DP human thymocytes on thymic lobes of mice deficient in MHC classes I and II (referred to herein as MHC-deficient mice) or WT mice for 72 hours, and then examined expression of CD69 and CD5 as indicators of TCR engagement of MHC (33).…”

Distinct phases in the positive selection of CD8+T cells distinguished by intrathymic migration and T-cell receptor signaling patterns

“…We previously reported that steady-state cortical thymocytes expressing positive-selecting TCR transgenes migrate more rapidly than thymocytes bearing nonselecting or polyclonal TCRs (22,23). To resolve how the kinetics of migration evolves over time during positive selection, we examined thymocyte migration by two-photon microscopy at various times after introducing preselection DP thymocytes onto positiveselecting slices.…”

Stromal Cell Networks Regulate Thymocyte Migration and Dendritic Cell Behavior in the Thymus

“…Dynamic studies have shown that medullary thymic DCs are nonmotile (19) and exhibit stable bodies while extending their dendrites (13). However, to date, information on thymic DC localization regarding TSCs as well as on DC/TSC dynamic interactions is lacking.…”